Fig 2: PADI2 citrullinates RUNX2 via physical interaction.A 293 T cells were transfected with 3xHA-Runx2 together with or without Flag-PADI2 and cultured for 3 days after the transfection. Cells were lysed, immunoprecipitated with anti-HA antibody and protein G-conjugated magnetic beads, and immunoblotted with indicated antibodies. The red arrow indicates predicted citrullinated RUNX2. Co-IP experiment was performed in three independent experiments; the representative results are shown here. B Workflow showing the detection of citrullinated RUNX2 by Biotin-PG labeling (left). Recombinant human RUNX2 (rhRUNX2) isoform c (NP_004339) was in vitro citrullinated by rhPADI2. The samples were labeled with Biotin-PG and then separated by SDS-PAGE followed by transfer to PVDF membrane. The membrane was incubated with Streptavidin conjugated with horseradish peroxidase (Streptavidin-HRP) or was immunoblotted with anti-RUNX2 antibody (right). A red asterisk indicates citrullinated RUNX2. This experiment was performed in three independent experiments; the representative results are shown here. C In vitro citrullinated rhRUNX2 isoform c was analyzed by LC-MS/MS. The ten arginine (R) sites of rhRUNX2 citrullinated by PADI2 were identified (left column). The mouse RUNX2 isoform 1 was used for site-directed mutagenesis of the 10 R sites and, for this, the R site in mouse RUNX2 isoform 1 matching the corresponding each R site in hRUNX2 isoform c is shown in the right column.

Fig 3: Padi2 knockout mice exhibited reduced bone mass.A Physiognomy of Padi2 control (Cont), hetero (Het), and knockout (KO) litters at newborn and P7 (n = 4–6 in each group). B Representative micro-CT images of the distal femur at 4 months in the arterial view of a coronal section and cross-sectional views are shown for each genotype (scale bar:1 mm). C Histomorphometric analyses of 3D micro-CT data of control and Padi2 KO mice in both male (n = 9, Cont mice and n = 7, Padi2 KO mice) and female (n = 11, Cont mice and n = 12, Padi2 KO mice). BV/TV bone volume/tissue volume; Tb. Th trabecular thickness; Tb. N trabecular number; Tb. Sp trabecular separation. D, E Representative images of H&E stained distal femur and proximal tibiae from 4 months-aged control and Padi2 KO mice (scale bar: 500 μm, 200μm). F, G Representative images of immunohistochemistry (IHC) for PADI2 (F) and type 1 collagen (COL1) at 4 months-aged Cont and KO mice (G) (scale bar: 500 μm, 200 μm). H Representative images of TRAP-stained trabecular bone of distal femur from 4 months-aged Cont and KO mice. TRAP-positive purple spots indicate multinucleated osteoclasts (scale bar: 500 μm, 200 μm); We performed two or three independent experiments with three biological replicates for each group (D–H). I Osteoclast identification by TRAP staining. Bone marrow-derived macrophages (BMMs) isolated from Cont and KO mice were differentiated into osteoclasts in the presence of CSF1 (20 ng/ml) and RANKL (100 ng/ml) for 5 days (scale bar: 10 μm). The graph shows the number of TRAP-positive osteoclasts with more than three nuclei (n = 8 in each group). Three independent experiments with eight biological replicates for each group. Data are expressed as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N.S, not significant.

Fig 4: PADI2-catalyzed citrullination of RUNX2 is required for RUNX2 stabilization.A MC3T3-E1 cells were transfected with empty vector (EV), Strep-Runx2 wild type (Wt), or R-to-K mutants and cultured in osteogenic media for 3 days after transfection. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The arrow indicates non-specific bands (n.s). Western blot data were collected from three independent experiments; the representative results are shown here. B, C pOB cells and hMSCs were transfected with EV, Strep-Runx2 WT, or R381K mutant plasmids and cultured in osteogenic media for 2 days. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified and normalized with β-Actin using ImageJ software. Western blot data were collected from two independent experiments; the representative results are shown here. D 293 T cells were transfected with Strep-Runx2 Wt or R-to-K mutants with or without Myc-Cbfβ plasmids and then incubated for 3 days after transfection. Cells were lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with indicated antibodies. Co-IP experiment was performed in three independent experiments; the representative results are shown here. E MC3T3-E1 cells were transfected with Strep-Runx2 Wt or R-to-K mutants and cultured for 2 days after transfection. Cells were fixed with 4% PFA, permeabilized, and then immunofluorescent staining was performed using anti-Strep-Tag II antibody. DAPI was used for the nucleus. Three independent experiments were performed and the representative results are shown here. Scale bar, 20 μm.

Fig 5: PAD2 and PAD4 reduce P. aeruginosa (PGO2330) biofilm biomass and viability. a Live/dead staining of PGO2330 biofilm with SYTO9 (left column), propidium iodide (PI) (middle column), and merged image (right column) at 24 h with rPAD2 (middle row) or rPAD4 (bottom row). Images captured at ×40 magnification. b, c Quantification of the average biofilm biomass (b) and thickness (c) was performed using COMSTAT2.1. Data were expressed relative to the control. d Mean PI fluorescence was quantified using ImageJ. Data were analysed by a one-way ANOVA followed by multiple comparison Tukey’s post hoc test and presented as mean ± SEM; n = 5 biological replicates. SEM, standard error of the mean.